Methods using polarimetry, colorimetry, reductimetry and a variety of chromatographies have been developed as methods for quantitative analysis of sugars such as sucrose and glucose. These methods, however, are all poorly specific to sugars and hence have poor accuracy. Among these methods, the polarimetry is simple in manipulation, but is largely affected by the temperature during the manipulation. Therefore, the polarimetry is not suitable as a method of simple quantification of sugars at home, etc. for ordinary people.
Recently, various types of biosensors utilizing the specific catalytic action of enzymes have been under development.
The following will describe a method of glucose quantification as one example of the method of quantifying a substrate contained in a sample. As an electrochemical method of glucose quantification, a method using glucose oxidase (EC 1.1.3.4: hereinafter abbreviated to GOD) as an enzyme and an oxygen electrode or a hydrogen peroxide electrode is generally well-known (see “Biosensor” ed. by Shuichi Suzuki, Kodansha, for example).
GOD selectively oxidizes β-D-glucose as a substrate to D-glucono-δ-lactone using oxygen as an electron mediator. In the oxidation reaction process by GOD in the presence of oxygen, oxygen is reduced to hydrogen peroxide. The decreased amount of oxygen is measured by the oxygen electrode, or the increased amount of hydrogen peroxide is measured by the hydrogen peroxide electrode. Since the decreased amount of oxygen and the increased amount of hydrogen peroxide are proportional to the content of glucose in the sample, glucose quantification is possible based on the decreased amount of oxygen or the increased amount of hydrogen peroxide.
The above method utilizes the specificity of enzyme reactions to enable accurate quantification of glucose in the sample. However, as speculated from the reaction process, it has a drawback in that the measurement is largely affected by the oxygen concentration of the sample, and if the oxygen is absent in the sample, the measurement is infeasible.
Under such circumstances, glucose sensors of new type have been developed which use as the electron mediator potassium ferricyanide, an organic compound or a metal complex such as a ferrocene derivative and a quinone derivative without using oxygen as the electron mediator. In the sensors of this type, the reduced form of the electron mediator which results from the enzyme reaction is oxidized on a working electrode, and based on the amount of this oxidation current, the concentration of glucose contained in the sample can be determined. Simultaneously, on a counter electrode, a reaction in which the oxidized form of the electron mediator is reduced into the reduced form of the electron mediator proceeds. With the use of such an organic compound or metal complex as the electron mediator in place of oxygen, it is possible to form a reagent layer comprising a known amount of GOD and the electron mediator which are carried in a stable state and a precise manner on the electrodes, so that accurate quantification of glucose is possible without being affected by the oxygen concentration of the sample. In this case, it is also possible to integrate the reagent layer containing the enzyme and electron mediator, in an almost dry state, with an electrode system, and hence disposable glucose sensors based on this technique have recently been receiving a lot of attention. A typical example thereof is a biosensor disclosed in Japanese Patent Publication No. 2517153. With such a disposable glucose sensor, by simply introducing a sample into the sensor connected detachably to a measurement device, glucose concentration can be measured readily by the measurement device.
According to the measurement method using the above-described glucose sensor, with the use of a sample whose amount is in the order of several μl, the concentration of a substrate in the sample can be determined readily. However, in recent years, it is anxiously desired in various fields to develop biosensors capable of measuring a sample in an extremely small amount of not more than 1 μl. When a sample in an extremely small amount is measured by a conventional electrochemical glucose sensor, the amount of glucose in the sample is also extremely small, and hence the sensitivity of the measurement may lower in some cases.
Thus, there has been developed a biosensor which utilizes two substantially comb-shaped electrodes comprising a plurality of branches that are arranged alternately on a base plate. FIG. 7 is a sectional view of the biosensor in the vicinity of the electrode system. In this type of sensor, the oxidized form of an electron mediator, which results from the oxidation at a first electrode 1 disposed on a base plate 5, can be reduced back to the reduced form at a neighboring second electrode 3, and the reduced form can be oxidized again at a neighboring first electrode 1. Accordingly, the value of the current flowing through the first electrode 1 increases apparently, so that more sensitive glucose quantification is possible in comparison with conventional biosensors.
This approach is applicable not only to glucose quantification but also to quantification of other substrates contained in a sample.
However, since there has been a demand in recent years for further reduction in the amount of a sample necessary for making a measurement, glucose sensors having higher sensitivity are anxiously desired in various fields.
In view of the above, an object of the present invention is to provide a highly sensitive biosensor capable of giving good response even when the amount of a sample is extremely small.